Human Aldo-Keto Reductases (AKR1A1, AKR1C1-AKR1C4) catalyze the activation of polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to yield catechols which autooxidize to electrophilic and redox-active PAH o-quinones. In human lung adenocarcinoma (A549) cells this pathway results in the production of reactive oxygen species (ROS) and formation of the mutagenic lesion 8-oxo-2'-deoxyguanosine (8-oxo-dGuo). Thus, in addition to PAH diol-epoxide-DNA adducts, oxidative DNA lesions may contribute to lung cancer initiation. Recently, AKR1C1-1C3 and AKR1B10 genes were identified as tobacco exposure and response genes in human bronchial epithelial and buccal cells. Four aims will elaborate the role of the AKR pathway in cancer of the airway. In Aim#1, the phase I [quinone reductases (QR)] enzymes that redox-cycle the PAH o-quinones will be identified. Unexpectedly, AKRs have substantial PAH o-quinone reductase activity and we will compare their catalytic efficiency and ability to generate ROS with NQO1. The phase II enzymes that remove the PAH- catechols (COMT, SULTs and UGTs) and PAH o-quinones (GSTs) from this redox-cycle will also be identified in vitro. In Aim#2, the metabolic fate of the PAH o-quinone ([3H]-benzo[a]pyrene-(B[a]P)-7,8-dione) will be elucidated in human bronchoalveolar (H358), A549 and immortalized bronchial epithelial (HBEC-tk) cells that produce this metabolite. Catechol and o-quinone conjugates will be identified and enzymes responsible phenotyped using real-time qRT-PCR. Aims 1 and 2 will identify enzymes whose polymorphic variants may affect individual susceptibility to PAH exposure and response. Aim #3, will determine whether the aryl hydrocarbon receptor (AhR) delivers PAH o-quinones to the nucleus to cause oxidative DNA damage. Studies will be performed in murine Hepa1 cells and their genetic AhR and ARNT negative variants and in HBEC-tk cells in which either AhR or ARNT have been silenced with si-RNA. The formation of 8-oxo-dGuo will be measured by an immunoaffinity capture LC-MS stable isotope dilution assay. Aim#4 will determine whether cigarette smoke condensate alters the transcriptome in HBEC-tk cells and oral dsyplasia cells with consequential elevation of AKR expression and 8-oxo-dGuo formation upon exposure to B[a]P or B[a]P-7,8- trans-dihydrodiol. These studies will inform genotyping and validate genomic studies in lung and buccal cancer. They will demonstrate that by acting as cigarette smoke and response genes, AKRs increase oxidative DNA damage and the mutagenic burden.